JP2013085927A - Method for coagulating blood that has flown out and method for using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To rapidly coagulate blood that has flown out from a living tissue.SOLUTION: The surface of blood that has flown out from the living tissue is irradiated with light of a blue-violet spectral bandwidth with the wavelength of 380-515 nm, that is emitted from a light emitting diode (LED).

Description

  The present invention relates to a method for coagulating blood flowing out from a living body, which is useful in fields such as medicine, dentistry, and biology.

  In recent years, medical and dentistry treatment technologies have been remarkably developed. However, even if the technology is developed, bleeding that occurs when a living tissue is invaded is an inevitable phenomenon. Similarly, bleeding occurs whenever a living body is wounded for other reasons. Depending on the scale of the bleeding treatment, if bleeding is to the extent that capillaries have been cut, a method of pressing the bleeding part so far and stopping with a blood coagulation system originally possessed by a living body such as platelets A method of burning a bleeding part and heat coagulating a living tissue or a method of suppressing bleeding with a drug has been taken. Recently, various types of laser hemostasis techniques have been developed. However, there is still a need for a better treatment method that does not place a burden on the living body and a method for quickly stopping hemostasis.

  Japan has an aging society and the average life expectancy is the highest in the world. People's hope is becoming more focused on the quality of life (QOL) of living better than just prolonging life, and speaking and eating are important functions that lead to life, especially for the elderly. In that sense, maintaining the health of the masticatory organs, including the preservation of teeth, can be said to be a major factor affecting QOL. Mastication is an indispensable function for food intake, and in recent masticatory system research, mastication stimulates brain cells to promote mental and neural development and activation, enhances immune function, and further improves obesity. The effects on various systemic functions such as suppression are becoming clear. Therefore, the deterioration of the masticatory function due to the loss of teeth leads to the possibility of accelerating the path to dementia and causing lifestyle-related diseases. The proportion of the population currently suffering from periodontal disease exceeds 80% in the middle-aged and older generation of 35 to 65 years old, and the demands of people in periodontal treatment are high and the contents are various. In addition, many related factors are involved in the onset and progression of periodontal disease, and cannot be solved only by preventive measures centered on oral cleaning. Unfortunately, many of the treatments for periodontal disease require recovery of function and aesthetic improvement. At present, these demands are mainly focused on non-regenerative therapies that have been used in the past. Therefore, there is a need for an innovative treatment that can regenerate lost periodontal tissue.

  Teeth penetrate the epithelium and are exposed in the oral cavity, and the continuity of the epithelium is lost at the boundary between the teeth and the gingiva, and it is in a very special environment in the living body. Teeth and gingiva are composed of “epithelial attachment” and “connective tissue attachment”. In the former, the epithelium called the junctional epithelium adheres to the tooth surface (enamel) via the hemidesmosome and the basement plate. The latter is composed of periodontal ligament (periodontal ligament), collagen fibers are embedded in the cementum of the periodontal surface while being calcified, and the fibers are also embedded in the alveolar bone while being calcified, and transferred to gingival fibers. Thus, the teeth are firmly bonded to the alveolar bone and gingiva.

  Periodontal disease is an inflammatory disease in the periodontal tissue caused by plaque bacteria, and is classified into “gingivitis” and “periodontitis”. “Gingivitis” in which inflammation is limited only to the gums, and when the inflammation is in the periodontal ligament, alveolar bone, or the attachment by the periodontal ligament is destroyed, is called “periodontitis”. Usually it progresses from gingivitis to periodontitis, forming pockets (grooves) around the teeth. As the pocket gets deeper, plaque bacteria in the pocket grow and inflammation progresses deeper. Ambiguity seen when systemic modifiers (drugs, blood disorders, immune disorders, nutritional status, stress, fatigue, smoking, etc.) are involved, and when the mechanical burden on teeth such as tooth decay is excessive Local remediation factors called trauma exacerbate inflammation. In cases with severe periodontitis and highly advanced tissue destruction, the periodontal tissue that was once lost even after evacuation was not restored to its original morphological function, and significant functional / esthetic disorders were observed after healing. However, this is a major factor that lowers the QOL of patients.

  Alveolar bone resorption is one of the characteristic pathologies of periodontitis, but the loss of periodontal cementum and periodontal ligament (periodontal ligament) that occurs at the same time is the essential condition of periodontitis. As the name suggests, the periodontal ligament has a ligament-like structure, and the tooth is suspended in the socket of the alveolar bone via the cementum on the root surface, acting as a cushion against strong occlusal pressure, and also rich in vascular components and metabolic activity It plays an important role in maintaining periodontal homeostasis. Despite numerous studies aimed at periodontal tissue regeneration for nearly half a century, it is very difficult to simultaneously regenerate this periodontal ligament tissue as well as the alveolar bone. That's why. When periodontal ligament regeneration is not accompanied, it has been clarified that entry into the defect of the oral epithelium and bone adhesion of the roots occur and heal in a biologically unstable state. It may cause a recurrence of the flame and loss of teeth.

  In order to treat this periodontitis, scaling and root planing (S / RP) is currently performed. This is the basic method of treatment for periodontitis, infectious tissue is mechanically removed at the site affected by periodontitis, and the root surface cementum and roots contaminated mainly by bacterial infection. This is a method that expects natural healing by removing the membrane and gingival tissue using a special instrument, giving the root surface a form in which surrounding tissue is easily attached. The healing form mainly consists of epithelial adhesion formed by epithelial progression and proliferation on the treatment root surface. This method is a necessary and sufficient treatment method as long as it does not require surgical treatment, can be treated only with no anesthesia or local infiltration anesthesia, and has no aesthetic and functional problems. However, in cases where alveolar bone is destroyed in advanced periodontitis, this procedure is not sufficient if reinfection is expected after treatment. In addition, a surgical procedure is likely to be difficult at a site where the tooth shape is complicated, and in this case, a surgical treatment method is applied. Surgical procedures such as surgically forming a gingival flap (flap) and treating under clear vision are performed on a site having a complicated shape that cannot be treated only with S / RP. The healing form basically expects the same epithelial adhesion as S · RP. And these treatments always need to suppress the phenomenon of bleeding.

Under such circumstances, regenerative medical technology for actively reconstructing periodontal tissue has been actively researched. Some techniques have already been used for treatment, and include bone resection, bone transplantation, mucosal transplantation and the like. This alveolar bone graft is classified into autologous, other family, heterogeneous bone graft, and artificial bone graft.
This is a method in which autologous bone transplantation is mainly performed, and a bone fragment collected from another site in the oral cavity is transplanted into a bone defect part. In allogeneic bone transplantation, decalcified freeze-dried allograft bone (DFDBA) has been used in Europe and the United States, and has achieved good clinical results. A representative artificial bone is hydroxyabatite (HA). Compared to S / RP alone, the healing form also works effectively in maintaining clots at the defect site, and it is thought that it can provide conditions that prevent periodontal ligament cells from migrating at the same time as inhibiting epithelial invasion. Have been reported that the main form of healing is epithelial adhesion that does not cause regeneration of the new cementum and periodontal ligament. Autologous bone grafts have the lowest antigenicity and infection problems and are naturally accepted by the receiving department. In addition, there is an advantage that the bone grafting material can be expected to have osteoconductivity and osteoinductivity. However, it is difficult to secure a necessary amount of transplanted bone by autologous bone transplantation. In addition, allogeneic and xenogeneic bone transplantation still has problems of antigenicity and infectivity. Regardless of the type of grafting material, if the bone defect site is large and the area where the root surface and the transplanted bone are in contact is large, There was a problem of causing adhesion (ankylossis).

  In the late 1970s and 1980s, a Scandinavian research group confirmed that epithelial tissue and gingival connective tissue lack the ability to regenerate periodontal ligaments, and that bone tissue causes bone adhesion to the root surface. It has been found that for the regeneration of the tissue, cells derived from periodontal ligament tissue must be present on the defect part, particularly on the root surface. The tissue regeneration induction method (GTR method) was devised based on this concept. This method suppresses epithelial cells that have a rapid growth rate in the healing process and rapidly enter the defect with a biocompatible blocking membrane. At the same time, the periodontal ligament tissue can be regenerated by space-making the defect. This method is currently the method that can be expected to regenerate periodontal tissue most, and has achieved good results at a site showing a bone defect form that is easy to make space. However, with this method, the clinical procedure is complicated, and it is difficult to accurately place a biocompatible film on the root surface for sites that show complex defects. The membrane had to be completely covered with a gingival flap, and the prognosis was easily affected by the surgical procedure and the environment. In addition, the biocompatible membrane has absorbability and non-absorbability, and when the former is used, re-operation is necessary to remove the membrane, and the latter does not require re-operation, but space making is necessary. Problems remain in strength. In addition to expecting cell migration from the remaining periodontal ligament, the amount of regeneration is limited, and from the viewpoint of membrane retention, the indication is only a part of vertical bone defect. Horizontal bone resorption is the main pathological condition in the widespread chronic periodontitis that is widely seen in middle-aged and elderly people, and there are many cases that this GTR cannot cope with.

  Thus, even though it is known that periodontal ligament-derived cells are an indispensable factor in the regeneration of periodontal tissue, conventional therapies such as this GTR method have been applied to growth factors, but have been applied from residual tissues. Only cell migration is expected, so the amount of tissue regeneration is greatly influenced by the defect form or the amount of residual periodontal ligament tissue, and its indication is limited at present. In highly destroyed periodontal tissues, it is becoming clear that it is not sufficient to expect cells that are the basis for regeneration from migrating from the remaining surrounding tissues, and it is necessary to supply them exogenously. The purpose of regenerative therapy in periodontal tissue is focused on how to suppress this epithelial healing and acquire connective tissue adherence by the periodontal ligament.

  In recent years, several studies aiming at tissue regeneration by performing the cell transplantation method have been reported. In many cases, single cells are seeded in a three-dimensional matrix and implanted and transplanted into a tissue defect, but none of them has yet achieved the expected tissue construction. The reason may be that it is difficult to select the cell source and control the localization of cell differentiation within the tissue defect. It is essential for the regeneration of periodontal tissue to be accompanied by the formation of cementum on the root surface, and it is necessary to simultaneously regenerate and functionally connect not only the soft tissue of the periodontal ligament but also the cementum and the hard tissue of the alveolar bone. There is. If these tissues are formed with a time difference due to the action of different tissue-specific progenitor cells and growth factors, cell transplantation in periodontal tissue regeneration must be more delicate. In other words, instead of simply injecting a single cell into a space-made defect and entrusting it to tissue differentiation in vivo, it is necessary to define the location of the cell and place an appropriate cell at each site. Is done.

  The cells required in this case have been cultured on the surface of glass or on the surface of synthetic polymers subjected to various treatments. For example, various containers and the like subjected to surface treatment using polystyrene as a material, for example, γ-ray irradiation, silicone coating, and the like are widely used as cell culture containers. Cells cultured and proliferated using such a cell culture container are peeled and collected from the container surface by treatment with a protease such as trypsin or a chemical. However, when recovering cells grown by chemical treatment as described above, the treatment process becomes complicated, the possibility of contamination is increased, and the grown cells are altered or damaged by chemical treatment. Disadvantages have been pointed out that there are cases where the original function of cells is impaired.

  In order to overcome such drawbacks, several techniques have been proposed so far. Among them, in Patent Document 1, in particular, an anterior ocular segment-related cell is cultured on a cell culture support having a substrate surface coated with a temperature-responsive polymer having an upper or lower critical solution temperature of 0 to 80 ° C. If necessary, the cultured cell layer is layered by a conventional method, and the cultured cell sheet is peeled off only by changing the temperature of the support, thereby making it possible to produce a cell sheet having sufficient strength. In addition, this cell sheet also retains a basement membrane-like protein, and the engraftment to the tissue is clearly improved as compared with the above-mentioned dispase-treated one. Then, periodontal ligament cells are cultured on a cell culture support whose surface is coated with the temperature-responsive polymer to obtain a periodontal ligament cell sheet, and then the culture solution temperature is equal to or higher than the upper critical solution temperature or the lower critical solution temperature. In vitro, the cultured periodontal ligament cell sheet is closely attached to the polymer membrane, and is peeled off together with the polymer membrane, and is three-dimensionally structured by a predetermined method, thereby reducing structural defects in vitro. It has been found that a cell sheet having several functions as a periodontal ligament-like tissue and a three-dimensional structure are constructed (Patent Document 2, Non-Patent Document 1, and Non-Patent Document 2). However, in any of the methods, in order to better regenerate the living tissue, it is necessary to study for better adhesion to the living body.

JP-A-2-21865 No. 2005-103233

Tissue Engineering, 11 (3/4), 469-478 (2005) Journal of Periodical Research, 43, 364-371 (2008)

  The present invention has been made with the intention of solving the above-described problems relating to coagulation of blood flowing out of a living body. Specifically, as a method of suppressing bleeding from the living body, a method of amorphizing and coagulating the surface of the flowing blood is shown. In addition, it was made with the intention of proposing a method of using the coagulation method found in the process. That is, an object of the present invention is to provide a method for coagulating blood flowing out of a living body from an idea completely different from the prior art. Another object of the present invention is to provide a method for using the coagulation method.

  In order to solve the above-mentioned problems, the present inventors have studied and developed from various angles. As a result, when light in the blue-violet spectral band having a wavelength of 380 to 515 nm emitted from a light emitting diode (LED) is irradiated to blood that has flowed out of the living tissue, the blood surface is rapidly coagulated and has an effect on the living body. Was found to be very low. Moreover, the coagulation method not only has an effect of stopping bleeding, but also has been found to be extremely useful as a place for regenerating a living tissue or a place for adhesion of a periodontal ligament cell sheet for the purpose of tissue regeneration. . The present invention has been completed based on such findings.

  That is, the present invention provides a method of irradiating light in a blue-violet spectral band having a wavelength of 380 to 515 nm, which is emitted from a light emitting diode (LED), as a method of coagulating blood flowing out of a living tissue. Further, the present invention is a method of using a blood clot useful for tissue regeneration obtained by the blood coagulation method, and additionally transplanting a cell sheet effective for living tissue regeneration medicine to the blood coagulation direction obtained by the coagulation method. Provide a method to use as a place.

  According to the method of the present invention, the surface of blood that has flowed out of a living tissue can be rapidly coagulated. This method not only suppresses bleeding, but is also useful as a place where blood clots obtained by coagulation regenerate living tissue and adhere cell sheets. Such a technique is useful at any site in a living body, and the present invention is a very useful invention in the fields of medicine, dentistry, biology and the like such as cell engineering and medical engineering.

  It is a figure which shows the appearance of the bleeding of the affected part immediately after tooth extraction of Example 1. FIG.   It is a figure which shows the appearance after irradiating the blue LED light of Example 1 for 10 seconds.   It is a figure which shows the appearance of the affected part one week after Example 1. FIG.   FIG. 4 is a diagram showing an experimental method of Example 2.   It is a figure which shows the result of having observed the cross section of the blood coagulated in Example 2 with the transmission electron microscope. The portion indicated by the arrow (→) in the figure is an amorphous layer on the blood surface formed by blue LED irradiation, and has the role of suppressing blood flowing out of the living tissue due to the presence of this layer. Small * marks in the figure are platelets collected by the formation of the amorphous layer, and large * marks are other cell components collected by the formation of the amorphous layer. PML in the figure indicates polymorphonuclear leukocytes, and RBC indicates erythrocytes. The bar in the figure indicates 2 μm.   It is a figure which shows the result of having expanded the arrow part of FIG. 5 of Example 2, and having observed with the transmission electron microscope. As in FIG. 5, the portion indicated by the arrow (→) in the drawing is an amorphous layer on the blood surface formed by blue LED irradiation, and has the role of suppressing blood that has flowed out of the living tissue due to the presence of this layer. Small * marks in the figure are platelets collected by the formation of the amorphous layer, and large * marks are other cell components collected by the formation of the amorphous layer. RBC in the figure indicates red blood cells. The bar in the figure indicates 1 μm.   It is a figure which shows the result of having observed the appearance of the blood at the time of blue LED non-irradiation of the comparative example 1 with the transmission electron microscope. No amorphous layer was observed on the blood surface, and no platelets were observed to collect. RBC in the figure indicates red blood cells. The bar in the figure indicates 1 μm. FIG.

  The present invention is a technique for coagulating blood flowing out of a living tissue. The method is based on irradiating the blood surface with light emitted from a light emitting diode (LED) in a blue-violet spectral band having a wavelength of 380 to 515 nm. When this blue LED was irradiated to the blood surface, it reacted with hemoglobin (maximum absorption wavelength: 430 nm), and it was found that the blood surface immediately coagulates and can be stopped. The present invention uses light emitted from a blue LED including the 430 nm region, and is not particularly limited as long as this light is used. A diode, apparatus, and irradiation jig for generating the light are not particularly limited. It is not limited to the kind, size, shape and the like. In the present invention, the irradiation time of the blue LED light, the irradiation distance, and other irradiation methods are not limited at all. Utilizing the light emitted from the blue LED of the present invention is advantageous without generation of ultraviolet rays harmful to living tissue.

  Although the blood coagulation mechanism according to the present invention has not been fully elucidated, when the light emitted from the blue LED of the present invention is irradiated to the blood flowing out of the living tissue, the blood surface becomes amorphous and a thin layer is formed. It was found that this surface layer confined blood components. In the present invention, the blood confined by the amorphized surface layer is referred to as a blood clot, and this blood clot also indicates that it is extremely effective for the regeneration of living tissue. There are no particular limitations on the use of such clots. For example, it is extremely useful in the process of healing wounds, ulcers, etc. in various parts of the body, and in the preparation of cell sheet transplants for the purpose of tissue regeneration. It is. It is also very useful as a place to transplant periodontal ligament cell sheets for the purpose of dental regeneration, gum regeneration after extraction, tissue regeneration induction treatment (GTR method), implant fixation, periodontal tissue regeneration It is. Furthermore, the method of the present invention is extremely useful as a hemostatic means having little influence on living tissues when, for example, a patient taking warfarin or the like and treating a patient whose blood is hard to solidify.

  Here, the periodontal ligament cell sheet used in the present invention is a ligament-like microstructure that is in close contact with a carrier ("ligament-like" is a state in which the cultured periodontal ligament cell sheet is in close contact with and oriented to the periodontal dentin Is a cultured periodontal ligament cell sheet. Periodontal ligament fibroblasts as suitable cells used for the preparation of the cultured periodontal ligament cell sheet of the present invention, or the periodontal ligament fibroblasts, cement blast cells, osteoblasts, gingival fibroblasts, vascular endothelial cells, Examples include a mixture of at least one or more mesenchymal stem cells, but the type is not limited. Periodontal ligament cells as used herein include periodontal ligament fibroblasts, cementoblasts, osteoblasts, gingival fibroblasts, vascular endothelial cells, mesenchymal stem cells, and their stem cells. Think. In the present invention, the highly engrafted regenerated periodontal ligament cell sheet means a sheet in which the various cells described above are cultured in a single layer on a culture support and then peeled off from the support. The obtained cell sheet has a lower side in contact with the culture support at the time of culture and an upper side opposite to the lower side. When culturing cells, if a cell culture support having a substrate surface coated with a temperature-responsive polymer having an upper or lower critical solution temperature of 0 to 80 ° C. in water according to the present invention is used, There are abundant adhesion proteins produced by the cells themselves during culture.

  The highly engraftment regenerated periodontal ligament cell sheet of the present invention is the periodontal fibroblasts described above, or the periodontal fibroblasts, cement blasts, osteoblasts, gingival fibroblasts, vascular endothelial cells, It may be a single-layer sheet in which at least one or more cells of leaf stem cells are mixed, or may be a sheet obtained by laminating them. Here, the laminated sheet may be a state in which the highly engrafted regenerated periodontal ligament cell sheet alone or in combination with a sheet made of another cell, such as the periodontal fibroblasts described above or the periodontal ligament thereof. A cell sheet in which at least one cell of at least one of cement blast, osteoblast, gingival fibroblast, vascular endothelial cell, and mesenchymal stem cell is mixed with fibroblast, and the above-described monolayer cell sheet In addition to this, there may be mentioned, but not limited to, a culture cell sheet composed of at least one cell of cement blast cells, osteoblasts, and gingival fibroblasts. Further, the position, order, and number of times of lamination are not particularly limited. For example, the periodontal fibroblasts described above, or the periodontal fibroblasts, cement blasts, osteoblasts, and gingival fibroblasts. The same cell sheet is laminated on the lower side or at least one or both of the upper side or the lower side of the monolayer cell sheet in which at least one of cells, vascular endothelial cells, and mesenchymal stem cells is mixed. A cell sheet composed of at least one cell of cement blast cells, osteoblasts, and gingival fibroblasts, which is separated from at least one or both of the lower side and upper side of the cell sheet, or a tooth root At least one of membrane fibroblasts, or periodontal fibroblasts, cement blasts, osteoblasts, gingival fibroblasts, vascular endothelial cells, and mesenchymal stem cells Even if the cell sheet consisting of at least one cell of the same cell sheet and another cement blast, osteoblast, gingival fibroblast is laminated to the monolayer cell sheet mixed with the above cells good. Further, the lamination is periodontal ligament fibroblast, or the periodontal ligament fibroblast is further added with cement blast, osteoblast, gingival fibroblast, vascular endothelial cell and mesenchymal stem cell. A cell sheet made of osteoblasts is stacked on the upper side of the mixed monolayer cell sheet, and a cell sheet made of cement blasts is stacked on the lower side, periodontal fibroblasts, or periodontal fibroblasts. Overlaying a cell sheet composed of gingival fibroblasts on top of a monolayer cell sheet mixed with at least one cell of cement blast cells, osteoblasts, gingival fibroblasts, vascular endothelial cells, mesenchymal stem cells, A cell sheet made of osteoblasts may be further stacked thereon, and a cell sheet made of cement blasts may be stacked on the lower side. The number of lamination is preferably 8 times or less, preferably 6 times or less, and more preferably 4 times or less. If it is more than 8 times, oxygen and nutrients will not reach the laminated cell sheet in the central part, resulting in cell death.

  The composition of the medium for culturing the above-described cells in the present invention is not particularly limited, and those usually used when culturing these cells may be used. For example, periodontal ligament fibroblasts, or cells in which at least one cell of cement blasts, osteoblasts, gingival fibroblasts, vascular endothelial cells, and mesenchymal stem cells is mixed with the periodontal ligament fibroblasts As a medium for cultivating the sheet, α-MEM medium, DMEM medium, or a mixture thereof mixed with 10% bovine serum, or further added with ascorbic acid diphosphate at a concentration of 50 μg / ml good.

  The highly engrafted cultured periodontal ligament cell sheet according to the present invention is not damaged by proteolytic enzymes such as dispase and trypsin during culture. Therefore, the highly engrafted cultured periodontal ligament cell sheet peeled from the substrate retains the cell-cell desmosome structure, has few structural defects, and has high strength. In addition, in the sheet of the present invention, the basement membrane-like protein between the cell and the substrate formed during the culture is not damaged by the enzyme. As a result, it is possible to adhere well to the affected tissue at the time of transplantation, and an efficient treatment can be performed. Specifically, when using a normal proteolytic enzyme such as trypsin, the cell-cell desmosome structure and the cell, the basement membrane-like protein between the substrate is hardly retained, Therefore, the cells are separated and separated in an individual state. Among them, dispase, a proteolytic enzyme, is known to be able to be detached in a state where the cell-cell desmosome structure is maintained at 10 to 60%. The obtained cell sheet is weak because it almost destroys the protein. In contrast, the cell sheet of the present invention is in a state where 80% or more of the desmosome structure and the basement membrane-like protein remain, and can obtain various effects as described above.

  The highly engrafted cultured periodontal ligament cell sheet in the present invention engrafts very well on the tooth root surface which is a living tissue. It has been found that this property is realized by suppressing the contraction of the cultured periodontal ligament cell sheet exfoliated from the support surface. At that time, the contraction rate of the cultured periodontal ligament cell sheet is desirably 20% or less, preferably 10% or less, and more preferably 5% or less in the length in any direction within the sheet. When the length in any direction of the sheet is 20% or more, the detached cell sheet sags, and even if it adheres to the living tissue in that state, it does not adhere to the tissue, and the high engraftment as shown in the present invention Sex cannot be expected.

  The method of not contracting the cultured periodontal ligament cell sheet is not limited as long as the method does not contract the cell sheet. For example, when the cultured periodontal ligament cell sheet is peeled off from the support, Examples include a method in which a ring-shaped carrier or the like cut out from a part is brought into close contact, and the cell sheet is peeled off together with the carrier.

  The carrier used when closely attaching the highly engrafted cultured periodontal ligament cell sheet is a structure for holding the cell sheet of the present invention so as not to contract, for example, a polymer film or a structure molded from the polymer film An object, a metallic jig, etc. can be used. For example, when a polymer is used as the carrier material, specific examples of the material include polyvinylidene difluoride (PVDF), polypropylene, polyethylene, cellulose and derivatives thereof, papers, chitin, chitosan, collagen, urethane, and the like. Can be mentioned.

  In the present invention, close contact refers to a state in which the cell sheet does not shift or move on the carrier at the boundary surface between the cell sheet and the carrier so that the cell sheet does not contract. Even if they are in close contact, they may be in close contact via a liquid (for example, a culture solution or other isotonic solution) existing between the two.

  The shape of the carrier is not particularly limited. For example, when transplanting the obtained highly engrafted cultured periodontal ligament cell sheet, a part of the carrier cut out to the same extent or larger than the transplant site When the cell sheet is used, the cell sheet is fixed only to the peripheral part of the cutout, and it is only necessary to apply the cell sheet in the cutout part to the transplantation site.

  Moreover, the high engraftment property on the tooth root surface, which is a feature of the cultured periodontal ligament cell sheet in the present invention, is realized under specific culture conditions. That is, the cultured periodontal ligament cell sheet of the present invention is obtained by seeding periodontal ligament cells on the surface of the support and then culturing. However, after the cells become confluent (full state) on the surface of the support 21 It has been found that it is preferably less than a day, preferably less than 15 days, and more preferably less than 10 days. If it is longer than 21 days, the activity of the cells in the lowermost layer of the cultured periodontal ligament cell sheet is reduced, so that the adhesion is also reduced, and the high engraftment shown in the present invention cannot be expected.

  The tooth root surface of the present invention is not particularly limited as long as it is a tooth root part, and generally includes an affected part in which a part or all of periodontal tissue is damaged or lost. The method of using the highly engrafted cultured periodontal ligament cell sheet of the present invention on such a root surface is not particularly limited, and examples thereof include a method of coating the highly engrafted cultured periodontal ligament cell sheet of the present invention. At that time, the cultured periodontal ligament cell sheet may be appropriately cut along the size and shape of the affected part. As described above, the highly engrafted cultured periodontal ligament cell sheet of the present invention can adhere to the tooth root surface, which is a living tissue, very well, and has never been obtained from the prior art.

  The temperature-responsive polymer used for coating the substrate in the cell culture support has an upper critical solution temperature or a lower critical solution temperature of 0 ° C to 80 ° C, more preferably 20 ° C to 50 ° C in an aqueous solution. If the upper critical lysis temperature or the lower critical lysis temperature exceeds 80 ° C., the cells may die, which is not preferable. Further, when the upper critical lysis temperature or the lower critical lysis temperature is lower than 0 ° C., the cell growth rate is generally extremely reduced or the cells are killed, which is also not preferable.

  The temperature-responsive polymer used in the present invention may be either a homopolymer or a copolymer. Examples of such a polymer include polymers described in JP-A-2-21865. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include a (meth) acrylamide compound, an N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative, or a vinyl ether derivative. Two or more of these can be used. Furthermore, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. Moreover, it is also possible to crosslink within a range that does not impair the original properties of the polymer.

  Examples of the base material to be coated include substances that can generally give form, such as glass, modified glass, polystyrene, polymethylmethacrylate, and the like, which are usually used for cell culture, such as polymers other than those described above. All compounds, ceramics, etc. can be used.

  The method for coating the support with the temperature-responsive polymer is not particularly limited, and may be, for example, the method described in JP-A-2-21865. That is, such coating is performed by applying a substrate and the above monomer or polymer to one of electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, organic polymerization reaction, or physical application such as coating and kneading. It can be performed by, for example, mechanical adsorption.

  The coating amount of the temperature-responsive polymer is preferably in the range of 0.5 to 5.0 μg / cm 2, preferably 1.0 to 4.0 μg / cm 2, more preferably 1.2 to 3.5 μg / cm 2. is there. When the coating amount is less than 0.5 μg / cm 2, the cells on the polymer are difficult to peel off even when a stimulus is applied, and the working efficiency is remarkably deteriorated. On the other hand, if it is 5.0 μg / cm 2 or more, it is difficult for cells to adhere to the region, and it becomes difficult to sufficiently attach the cells. The form of the support in the present invention is not particularly limited, and examples thereof include a dish, a multiplate, a flask, and a cell insert.

  In the present invention, cells are cultured on the cell culture support produced as described above. The culture medium temperature is not particularly limited as long as the polymer coated on the substrate surface has an upper critical solution temperature or lower, and when the polymer has a lower critical solution temperature or higher, the temperature is not particularly limited. However, it goes without saying that culturing in a low temperature range where cultured cells do not proliferate or in a high temperature range where cultured cells die is inappropriate. The culture conditions other than the temperature may be in accordance with conventional methods and are not particularly limited. For example, the medium to be used may be a medium to which serum such as known fetal calf serum (FCS) is added, or a serum-free medium to which such serum is not added.

  In the method of the present invention, in order to peel and recover the cultured cells from the support material, the cultured highly engrafted cultured periodontal ligament cell sheet is brought into close contact with the carrier, and the temperature of the support material to which the cells are attached is determined based on the support base. By making the temperature higher than the upper critical solution temperature or lower than the lower critical solution temperature of the coating polymer of the material, it can be peeled off together with the carrier. Note that peeling of the sheet can be performed in a culture solution in which cells are cultured or in another isotonic solution, and can be selected according to the purpose.

  In the present invention, after the cell sheet is applied to the affected area, the cell sheet may be peeled off from the carrier. The method of peeling is not limited at all, but for example, a method of wetting the carrier to weaken the adhesion between the carrier and the cell sheet, or using a knife such as a knife, scissors, laser light, plasma wave, etc. Also, a method of cutting may be used. For example, when using a cell sheet that is in close contact with a carrier that has been partially cut out as described above, cutting along the boundary line of the affected area using laser light or the like avoids attachment of the cell sheet to an extra area other than the affected area. It is convenient.

  The method for fixing the highly engrafted regenerated periodontal ligament cell sheet and the living tissue shown in the present invention is not particularly limited, and the cell sheet and the living tissue are joined and sutured with an adhesive that can be used in vivo. Alternatively, since the highly engrafted cultured periodontal ligament cell sheet as shown in the present invention is rapidly engrafted with the living tissue, it may be simply adhered to the affected area without using such means.

The lamination method of the laminated sheet in the present invention is not particularly limited, but the highly adherent cultured periodontal ligament cell sheet in close contact with the carrier described above may be performed by the following method.
(1) A cell sheet adhered to a carrier is adhered to a cell culture support, and then the medium is added to peel the carrier from the cell sheet, and further, a cell sheet adhered to another carrier is repeatedly adhered to the cell. A method of layering sheets.
(2) The cell sheet in close contact with the carrier is inverted and fixed on the carrier side on the cell culture support, another cell sheet is attached to the cell sheet side, and then the medium is added to peel off the carrier from the cell sheet. A method of layering cell sheets by repeating the operation of attaching another cell sheet.
(3) A method in which cell sheets in close contact with a carrier are brought into close contact with each other on the cell sheet side.
(4) A method in which a cell sheet in close contact with a carrier is applied to an affected part of a living body, the cell sheet is attached to a living tissue, the carrier is peeled off, and another cell sheet is stacked again.

  For the purpose of exfoliating and collecting the highly engrafted cultured periodontal ligament cell sheet with high yield, a method of gently tapping or shaking the cell culture support, or a method of stirring the medium using a pipette, etc. Or they may be used in combination. In addition, the cultured cells may be separated and recovered by washing with an isotonic solution or the like as necessary.

  Although the use of the highly engrafted cultured periodontal ligament cell sheet shown in the present invention is not limited at all, for example, degree periodontitis, severe periodontitis and other periodontal-related diseases, gingival recession, gingivitis, etc. Effective for gingival related diseases.

  According to the method of the present invention, the surface of blood that has flowed out of a living tissue can be rapidly coagulated. This method not only suppresses bleeding, but is also useful as a place where blood clots obtained by coagulation regenerate living tissue and adhere cell sheets. In addition, the highly engrafted cultured periodontal ligament cell sheet obtained by the above-described method is extremely superior in non-invasive points at the time of detachment as compared with those obtained by the conventional method. Clinical application is strongly expected as a membrane. In particular, unlike the conventional transplanted sheet, the highly engrafted cultured periodontal ligament cell sheet of the present invention has a high engraftability with the living tissue, and thus engrafts the living tissue very quickly. For this reason, GTR passively expected only cell migration from surrounding tissues (mainly the remaining periodontal ligament), but using this cell sheet allows transplantation of target cells at a very high density. become able to. Moreover, the problem of antigenicity and infectivity can be solved by using self cells. From the perspective of cell transplantation, research has been conducted on transplanting cells that have been three-dimensionally cultured in a collagen gel, and research on seeding and culturing cells on a bioabsorbable membrane and transplanting the entire membrane. However, the cell sheet is overwhelmingly superior in terms of cell density, and a cell sheet containing only pure cells / extracellular matrix components, which are free of inclusions such as absorbent membranes, is more advantageous for colonization in tissues. It is clear. Invasion of the epithelium during the healing process is a major issue, but it can be prevented if connective tissue cells have been established on the root surface in advance. In this colonization of cultured cells to the root surface, the extracellular matrix containing the adhesion molecules secreted by the cultured cells is collected and transplanted non-invasively at the same time as the cells. Expected to work favorably. The attached organs around the teeth have a special hierarchical structure such as cementum, periodontal ligament (ligament), and alveolar bone. It has been confirmed that the cells capable of constructing periodontal ligament tissue exist in the periodontal tissue only in the periodontal ligament and not in the alveolar bone or gingiva. There are various cells in the periodontal ligament (osteoblastic cells that form the supporting alveolar bone, fibroblasts that form the ligaments of the periodontal ligament, and cement blasts that form the cementum. However, tissue stem cells may naturally exist.) Research to isolate these cells is also underway, and cells collected from the periodontal ligament are prepared to produce cell sheets that are layered at the time of transplantation. It is thought that the adherent organ can be reconstructed more efficiently by giving a specific polarity. The above is considered to be an extremely effective technique for improving the treatment efficiency of the affected area and further reducing the burden on the patient.

  Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.

In the clinical field, with the consent of the patient, the extraction site was irradiated with light emitted from the blue LED. The apparatus and irradiation conditions used at that time are as follows.
1. Blue-violet LED 380-515 nm.
2. Power output at the light-guide tip: 942 mW.
3. Continuous output.
4). Light guide aperture: 1 cm in diameter (0.785 cm 2).
5. Output power measured on a power meter at 410 nm (Nova, Ophir, North Andover, Mass.) Showered proximity 750 mW / cm2 at a distance of 1 ft.
6). Irradiation area: approxmately 1.25 cm2.
7). Irradiation time of 10 sec quality energy 50 joules and an energy density of 7.5 J / cm2.
8). Irradiation time of 20 sec quality energy 100 joules and an energy density of 15 J / cm2.
9. Bluephase G2 device, Ivoclar Vivadent, Schaan, Liechtenstein
From the appearance of the affected area immediately after the extraction (FIG. 1), it can be seen that blood is flowing out from the living tissue. Thereafter, when the affected area was irradiated with blue LED light for 10 seconds, bleeding from the affected area stopped (FIG. 2). The appearance one week after the affected area is shown in FIG. From the above, it was found that the present invention has an effect of quickly stopping bleeding.

In order to examine the blood coagulation mechanism in the present invention, blood was placed on a Petri dish, irradiated with blue LED light (FIG. 4), and the obtained blood was observed with a transmission electron microscope. Samples to be observed with a transmission electron microscope were fixed with 2.5% glutaraldehyde (using 0.1 M phosphate buffer) for 2 hours, and then in 0.1 M phosphate buffer overnight at 4 ° C. It was obtained by immersing and further fixing with 1% OsO ₄ (using 0.1 M phosphate buffer) for 2 hours. FIG. 5 shows a cross section of the coagulated blood. It was found that the portion indicated by the arrow (→) in the figure is an amorphous layer on the blood surface formed by blue LED irradiation, and the blood flowing out from the living tissue is suppressed by the presence of this layer. Small * marks in the figure are platelets collected by the formation of the amorphous layer, and large * marks are other cell components collected by the formation of the amorphous layer. PML in the figure indicates polymorphonuclear leukocytes, and RBC indicates erythrocytes. It has been found that the amorphous layer formed by the blue LED light collects platelets and stops hemostasis more efficiently. FIG. 6 shows the result of observing with the transmission electron microscope by enlarging the arrow part of FIG. As in FIG. 5, the part indicated by the arrow (→) in the figure is an amorphous layer on the blood surface formed by blue LED irradiation, and it was found that the blood flowing out of the living tissue is suppressed by the presence of this layer. .

Comparative Example 1

The same experiment as in Example 2 was performed without irradiating the blue LED light. The appearance of blood at that time is shown in FIG. No amorphous layer was observed on the blood surface, and no platelets were observed to collect. RBC in the figure indicates red blood cells. The bar in the figure indicates 1 μm.
FIG.

On a commercially available 3.5 cmφ culture dish (Falcon 3001 manufactured by Becton Dickinson Labware), N-isopropylacrylamide monomer dissolved in isopropyl alcohol to 50% 0.07 ml was applied. An electron beam with an intensity of 0.25 MGy was irradiated to immobilize N-isopropylacrylamide polymer (PIPAAm) on the surface of the culture dish. After irradiation, the culture dish was washed with ion-exchanged water to remove residual monomer and PIPAAm not bound to the culture dish, dried in a clean bench, and sterilized with ethylene oxide gas to obtain a cell culture support material. . When the amount of the temperature-responsive polymer on the substrate surface was measured, it was found that 1.6 μg / cm ^{2 was} coated. Periodontal ligament tissue was collected from the maxillary molar portion of F344 nude rats, and periodontal ligament cells were collected by enzyme treatment according to a conventional method and seeded on the surface of each support material (1 × 10 ⁵ cells / 3. 5 cm Dish). As the medium, a DMEM medium supplemented with 10% bovine serum and ascorbic acid diphosphate so as to be 50 μg / ml was used. As a result of culturing at 37 ° C. and 5% CO ₂ , the periodontal ligament cells on the cell culture support material also adhered normally and proliferated. The cells cultured after 14 days of culture become confluent, and further, a medium molded from a polyvinylidene difluoride (PVDF) film with a diameter of 5 mm cut out into a circular shape with a diameter of 2 mm is placed on the cells cultured for 7 days, Was gently aspirated, incubated with the cell culture support material at 20 ° C. for 30 minutes and cooled, so that the cells on any of the cell culture support materials were peeled off with the carrier covered. The obtained cell sheet was sufficiently strong as a single sheet having a shrinkage rate of 5% or less.

  Next, a periodontal tissue defect part was created in the immunodeficient small animal, and blue LED light was irradiated to the affected part for 20 seconds. An attempt was made to transplant the cultured periodontal ligament cell sheet of Example 3 to the irradiated part. Specifically, bone defects were prepared in the maxillary molar mesial alveolar bone of F344 nude rats, the cementum was removed, and the root dentin was exposed to create experimental periodontal tissue defects. The lower part of the cultured periodontal ligament cell sheet of Example 3 was coated on the exposed dentin surface described above, and the transplant was completed by protecting the wound surface. One week after the operation, it was found that the cultured periodontal ligament cell sheet was in close contact with the root dentin and oriented, resulting in a ligament-like tissue. It was found that periodontal tissue could be reconstructed by transplanting the cultured periodontal ligament cell sheet of the present invention.

  According to the method of the present invention, the surface of blood that has flowed out of a living tissue can be rapidly coagulated. This method not only suppresses bleeding, but is also useful as a place where blood clots obtained by coagulation regenerate living tissue and adhere cell sheets. Such a technique is useful at any site in a living body, and the present invention is a very useful invention in the fields of medicine, dentistry, biology and the like such as cell engineering and medical engineering.

Claims (9)

A blood surface coagulation method that irradiates blood flowing out of a living tissue with light in a blue-violet spectral band having a wavelength of 380 to 515 nm emitted from a light emitting diode (LED) to coagulate the outflow blood surface. The blood surface coagulation method according to claim 1, wherein the coagulation method of the outflow blood surface is used for clot preparation. The blood surface coagulation method according to claim 1, wherein the coagulation method of the outflow blood surface is used for hemostasis. The blood surface coagulation method according to any one of claims 1 to 3, wherein the coagulation method of the outflowed blood surface is used for producing a transplanted surface of a cell sheet. The blood surface coagulation method according to any one of claims 1 to 4, wherein the coagulation method of the outflow blood surface is used for treatment of a dental region. The blood surface coagulation method according to claim 5, wherein the dental treatment is treatment for a patient taking warfarin. The blood surface coagulation method according to any one of claims 5 and 6, wherein the dental treatment is a tissue regeneration induction treatment method (GTR method). The blood surface coagulation method according to any one of claims 4 to 7, wherein the dental treatment is a periodontal ligament regeneration treatment using a periodontal ligament cell sheet. The blood surface coagulation method according to claim 8, wherein the periodontal ligament cell sheet is a laminate of cell sheets.